This thesis considers various roles of circadian clock genes in insect physiology. Application of molecular-biology methods in Pyrrhocoris apterus, non-model insect species, enable us to investigate involvement of circadian clock genes in photoperiod induced physiological responses. We discover involvement of neuroendocrine cells, and a role of Juvenile hormone (JH) signalization in transduction of photoperiodic signalization to peripheral tissues. We found new principles of JH signal diversification in tissue specific manner, and in addition described molecular mechanism of photoperiod induced changes in gut physiology. Comparison of gut and fat body tissue reveals that mechanism observed in the gut is tissue specific, and that circadian clock genes exhibit tissue specific functional pleiotropic effect.

Current models state that insect peripheral oscillators are directly responsive to light, while mammalian peripheral clock genes are coordinated by a master clock in the brain via intermediate factors, possibly hormonal. We show that the expression levels of two circadian clock genes, period (per) and Par Domain Protein 1 (Pdp1) in the peripheral tisue of an insect model species are inversely affected by contrasting photoperiods. The effect of photoperiod on per and Pdp1 mRNA levels was found to be mediated by the juvenile hormone. Our results provide the first experimental evidence for hormonal regulation of circadian clock gene expression in insects.

Our body is influenced by many cyclical changes in the environment, such as day and night or seasons. To predict these changes and react to them in time, the organism is equipped with inner clock, which rhythmically influences many physiological processes, such as sleep or metabolic rhythms. Disrupting our inner rhythms at molecular and behavioral levels contributes to many serious disorders. It is necessary that all mechanisms of the inner circadian clock are developed and set up properly. Circadian clocks are set up by the mother, who passes rhythmical information about day and night cycle on to her embryo. Though a great attention is devoted to revealing the nature of this synchronization between the mother and her pup, the mechanisms of this process have not been fully understood yet. The aim of this thesis is to contribute to actual understanding of this synchronization. Experiments, performed in this thesis, relate to studying the ability of maternal signals to synchronize embryos with the environment. Feeding and light regime of pregnant rats was manipulated and the effect of these changes on the neuronal activity within the suprachiasmatic nuclei of 19-day embryos was analyzed.

The circadian clock regulates a wide range of biological processes, allowing plants to be prepared for predictable daily diurnal changes in environmental cues such as light and temperature. Recent studies have suggested that the circadian clock may also control plant immunity. The exact nature of the interaction between the circadian clock and plant pathogens remains unknown. Our focus in this study is on the elucidation of the role of the biological clock in plant immunity against the necrotrophic pathogen to Botrytis cinerea. In order to do this we tested the level of susceptibility to B. cinerea in Arabidopsis thaliana wild type and transgenic plants: toc1, cca1/lhy, cca1/toc1, lhy/toc1, cca1/lhy/toc1, GLK1 OE, GLK2 OE, glk1, glk2, and glk1/glk2. We demonstrated that the time of infection plays a role in susceptibility to B. cinerea. Specifically, we found that plants are more susceptible to infection in the subjective morning. We also found that genetic mutations in core clock components or in GLK genes leads to changes in susceptibility to B. cinerea. Our data suggests that clock genes are not solely responsible for plant immune responses to B. cinerea but rather the ways in which the biological clock system regulates outcome pathways. Furthermore, when we entrain the biological clock by changing the photoperiod (day length) in normal earth conditions LD 24h and SD 24h, we observed that short day plants had higher susceptibility to B. cinerea than long day plants. In addition, when we entrain the biological clock in different photoperiods, the LD 30h photoperiod plants displayed similar responses as those in the SD 24h photoperiod. The data indicates that day length is not responsible for the control of plant immunity; it is the ability of light to entrain the biological clock that is important. Together, the data strongly support the conclusion that the circadian clock plays a role in plant defense regulation.

Les microalgues du phytoplancton sont exposées à des variations fréquentes et rapides de la qualité et de l'intensité spectrale en milieu marin. On peut donc supposer qu'il existe des mécanismes de photoperception spécifiques aux microalgues, différents de ceux identifiés chez les organismes terrestres. L'importance de l'horloge circadienne dans la transmission de l'information lumineuse et notamment la photopériode a largement été caractérisée chez plusieurs organismes modèles terrestres. Le principal objectif de ma thèse était d'étudier les régulations des gènes de l'horloge en réponse à la lumière, chez la microalgue Ostreococcus tauri. Le développement récent des techniques de génomique fonctionnelle chez cette microalgue eucaryote l'a promue comme un nouvel organisme modèle pour l'étude de mécanismes complexes tels que horloge circadienne. Mon étude s'est focalisée sur la caractérisation d'une voie de signalisation de type système à deux composants susceptible de transmettre le signal lumineux vers l'oscillateur central de l'horloge. J'ai étudié les régulations des principaux acteurs de l'horloge d'Ostreococcus par la lumière, et en particulier celles du gène TOC1. J'ai aussi caractérisé la protéine LOV-HK, un nouveau type de photorécepteur à la lumière bleue chez les eucaryotes, dont l'activité est requise pour le bon fonctionnement de l'horloge d'Ostreococcus. L'importance des régulations transcriptionnelles de TOC1 et de LOV-HK, ainsi que leurs fonctions dans l'oscillateur central ont été abordées par l'utilisation d'un promoteur inductible. Enfin, j'ai montré que LOV-HK et plus globalement l'horloge régulent la croissance cellulaire et la biomasse, démontrant leur rôle central dans le contrôle de la physiologie d'Ostreococcus tauri. / Light quality and intensity change frequently in the water column. Therefore marine microalgae are exposed to large changes in light spectrum. Photoperception mechanisms in microalgae are expected to differ from those of land plants since the marine environment has unique properties of light transmission. The focus of my PhD project concerns two mains topics, circadian clock regulation and photoperception in the microalga Ostreococcus tauri. In recent years, O. tauri has emerged as a promising model organism using functional genomics approaches to study complex processes such as the circadian clock regulations. My study was focused on the involvement of a two components system in light transmission to the circadian clock of Ostreococcus. I have studied the molecular mechanisms underlying the regulation of the core clock component TOC1. I have also characterized a novel eukaryotic blue light photoreceptor called LOV-HK, which regulates circadian clock function in Ostreococcus. Using an inducible promoter system to modulate the levels of TOC1 and LOV-HK, I have analyzed the importance of their transcriptional regulations in the clock. Finally, I have shown that LOV-HK and more generally the circadian clock, regulates cell growth and biomass in Ostreococcus tauri.

Die Acetyltransferase TIP60/KAT5 steuert die circadiane Uhr in Drosophila Endogene Uhren steuern verhaltensbezogene und physiologische Prozesse in einer rhythmischen Weise, die eine Periodizität von ~24 h aufweist. Drosophila zeigt einen robusten Rhythmus von Aktivitäts- /Ruhephasen, der von im Gehirn befindlichen zirkadianen Schrittmacherneuronen gesteuert wird. Angeborene Rhythmen der Genexpression werden durch positive und negative transkriptionelle/translationale Rückkopplungsschleifen, die die circadiane Uhr antreiben, generiert. Obwohl die Regulierung der Rückkopplung von Uhrengenen relativ gut verstanden ist, sind die Faktoren, die die Aktivatoren stimulieren, einen neuen circadianen Zyklus zu beginnen, noch unbekannt. Posttranslationale Modifikationen spezifischer Proteine durch Acetylierung erleichtert die Gentranskription. TIP60/KAT5 ist ein Mitglied der hochkonservierten MYST-Familie von Acetyltransferasen und ist an einer Vielzahl zellulärer Prozesse beteiligt, wie beispielsweise Zellwachstum und DNA-Reparatur, undzwar indem es verschiedene Zielproteine acetyliert. Das Hauptziel dieser Arbeit ist es, die Mechanismen, durch die Lysin-Acetyltransferase TIP60/KAT5 die circadiane Uhr von Drosophila steuert, zu untersuchen. Das binäre UAS/GAL4 System wurde eingesetzt, um einen gezielten knock down (durch RNA-Interferenz) oder eine Überexpression (eine Mutation in der für die Katalyse essentiellen Aminosäure, wodurch eine dominant negative Funktion verursacht wird) von TIP60/KAT5 in zirkadianen Schrittmacherneuronen zu erreichen. Das temperatursensitive GAL80 (TARGET) System wurde verwendet, um die normale Entwicklung der Schrittmacherneuronen bis ins Erwachsenenstadium zu ermöglichen. Unter Verwendung von nicht-invasiven lokomotorischen Aktivitätstests konnte gezeigt werden, dass TIP60 in Schrittmacherneuronen für den robusten zirkadianen Rhythmus in Abwesenheit von Zeitgebern im adulten Stadium erforderlich ist. In Übereinstimmung mit den beobachteten Veränderungen des circadianen Bewegungsverhaltens, war die rhythmische Expression von wichtigen clock-controlled genes wie Period und Timeless sowie Genen der Hilfsschleife Vrille und Clockwork orange gedämpft. Im Gegensatz dazu konnte gezeigt werden, dass die Tip60 mRNA unabhängig von der Uhr reguliert wird. Das zirkadiane Bewegungsverhalten wurde genutzt, um zu beurteilen, ob Untereinheiten des TIP60 (Nu4A) multimeren Komplexes auch an der Regulation des Rhythmus beteiligt sind. Um die Zielproteine von TIP60 in der circadianen Uhr zu bestimmen, wurde ein genetischer Interaktions-Test unter Verwendung des Phänotyps der circadianen Lokomotoraktivität durchgeführt und es wurde eine starke Interaktion mit dem zirkadianen Co-Aktivator CYCLE und dem Repressor PERIOD in trans-heterozygoten Kreuzungen gefunden. Aufgrund des Fehlens eines spezifischen Antikörpers, um die Funktion von TIP60 in vivo in adulter Drosophila zu untersuchen, wurde ein transgener Stamm (UAS dTIP60-HA) mit einem Hämagglutinin-Epitop-Tag erzeugt. Dieser transgene Stamm wurde genutzt, um TIP60-HA unter Verwendung eines ubiquitären Promotors (tub GAL4) überzuexprimieren. Ein Antikörper gegen das Epitop (Anti-HA) wurde verwendet, um einen Chromatin-Immunopräzipitations-Assay (ChIP) in diesen transgenen Fliegen durchzuführen. Es zeigte sich, dass TIP60 rhythmisch in der regulatorischen Region von clock-controlledgenes lokalisiert ist, was eine rhythmische Belegung von DNA-Elementen, ähnlich dem CLOCK/CYCLE Komplex, nahelegt. Co-Immunpräzipitation wurde mit transgenen Fliegen durchgeführt, um eine Protein-Protein-Wechselwirkung zwischen TIP60 und CYCLE nachzuweisen. Die Ergebnisse dieser Studie legen deutlich eine Beteiligung von TIP60/KAT5 als Co-Aktivator an der präzisen Steuerung der circadianen Uhr in Drosophila nahe. Deutsche Übersetzung: Dr. Inga Urban

Relógios endógenos controlam grande parte de processos biológicos através de osciladores bioquímicos que coordenam a sinalização de pistas ambientais até vias metabólicas, permitindo a percepção do tempo e adaptação a mudanças rítmicas. Comportamentos cíclicos diários foram primordialmente descritos em plantas e, mais recentemente, têm fornecido informações valiosas sobre os ciclos de retroalimentação da transcrição e tradução de genes que controlam estes osciladores. O florescimento é um exemplo bem conhecido da importância da percepção do comprimento do dia através do relógio, processo intimamente regulado por fotorreceptores e pelos genes centrais e periféricos do relógio biológico. Em organismos multicelulares há uma combinação específica de genes mais expressa em cada tecido, podendo ter funções, fases e períodos diferentes, o que aumenta a complexidade desse mecanismo. Devido a isso, tem-se buscado modelos alternativos mais simples dentro dos eucariotos fotossintetizantes relacionados às plantas terrestres. Modelos simplificados facilitam, por exemplo, a avaliação da combinação de fatores que induzem o estresse e como o relógio biológico se altera, permitindo a antecipação de mudanças ambientais e sincronização da fisiologia com o meio ambiente. Neste trabalho, verificou-se como o relógio circadiano se ajusta ao estresse em 3 diferentes modelos: Gracilaria tenuistipitata (Rhodophyta), Ostreococcus tauri (Chlorophyta) e Saccharum sp (Embryophyta). Para isso, estabeleceu-se em G. tenuistipitata métodos para avaliação de crescimento e da fluorescência da clorofila de modo automático, comprovando da existência de ritmos circadianos. Além disso, após padronização de genes de referência para normalização das RT-qPCRs, o gene TRX ficou superexpresso durante a primeira hora após o déficit hídrico. Já em O. tauri, onde os genes centrais do relógio são conhecidos, mudanças na expressão de LOV-HK e TOC1 estão relacionadas com maior crescimento em baixa e alta temperatura, respectivamente. Uma combinação específica de luz, temperatura e salinidade pode ser um importante indutor de eflorescências que reflete mudanças transcricionais no oscilador central, o que pode ser comparado às florescências de plantas terrestres. Já em Saccharum sp tolerante à seca, ritmos de fotossíntese e de expressão de CCA1 sofrem mudanças de fase em suas oscilações e transcritos de HVA-22 e DRP são significativamente mais expressos sob dessecação. Em suma, o estresse em Saccharum sp reseta o relógio, aumentando o período de oscilação da fotossíntese. Em O. tauri induz maior crescimento, mantendo as características do relógio. Não foi possível avaliar o efeito do estresse no relógio de G. tenuistipitata, mas ferramentas foram desenvolvidas visando este objetivo. Estudos de respostas do relógio podem fornecer informações valiosas para o entendimento da reprodução e crescimento de organismos com elevado potencial de aplicações biotecnológicas. / Endogenous clocks control a large range of biological processes through biochemical oscillators that coordinate the signaling of environmental cues to metabolic pathways, allowing the perception of time and adjust to rhythmic changes. Cyclical daily behaviors were first noticed in plants and, more recently, revealed information about the transcriptional-translational feedback loops of genes that control these oscillators. Flowering is a well-known process where the perception of day length by the clock is intimately regulated by photoreceptors and by the central and peripheric genes of the biological clock. Multicellular organisms have a tissue-specific combination of expressed clock genes that may have different phase and period, increasing the complexity of this mechanism. Due to this reason, alternative models have been proposed for land plants-related photosynthetic eukaryotes. New models can simplify, for example, which combination of factors induce stress and how the biological clock is altered, allowing the anticipation of environmental changes and synchronization of physiology and environmental factors. This work aimed to verify how the biological clock adjusts to different kinds of stresses in 3 species: Gracilaria tenuistipitata (Rhodophyta), Ostreococcus tauri (Chlorophyta) and Saccharum sp (Embryophyta). Automated measurement techniques for growth rate and photosynthesis were stablished for the red alga. This alga also showed, after establishment of reference genes for RT-qPCRs normalization, an overexpression of TRX during the first hour under water deficit. In O. tauri, where the central clock genes are known, changes in LOV-HK and TOC1 gene expression are related to a higher growth rate under low and high temperatures, respectively. Besides, a specific combination of light, temperature and salinity can be an important trigger of seasonal blooms that causes important transcriptional changes at the central oscillator, what is similar to land plants. In Saccharum sp tolerant to drought, photosynthesis rhythms and CCA1 expression change their phase under simulated water deficit and drought responsive transcripts like HVA-22 and DRP are significantly up-regulated. In short, stress resets the clock in Saccharum sp, increasing the period of photosynthesis oscillation. In O.tauri, it induces a higher growth, keeping clock features. It was not possible to verify clock responses to stress in G.tenuistipitata, but methods to do so were stablished. The biological clock responses to stress can provide invaluable information for the better understanding about the growth and reproduction of organisms with a high biotechnological potential

京都大学 / 新制・課程博士 / 博士(薬科学) / 甲第23142号 / 薬科博第141号 / 新制||薬科||15(附属図書館) / 京都大学大学院薬学研究科医薬創成情報科学専攻 / (主査)教授 土居 雅夫, 教授 中山 和久, 教授 竹島 浩 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

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Long-term exposure to constant light results in desynchronization of the circadian system in an adult and is associated with reduced efficiency of many physiological functions timed to the exact time of day, or with the development of some of the so-called civilization diseases. Constant light in adults also results in deterioration of the cognitive abilities or changes in the sleep structure. The effect of night light on the health of an adult organism is studied mainly in connection with shift work or with light pollution. The question of what effect the increased level of night light has on the development of the organism, especially on the development of the nervous system and the circadian system itself, is less studied. This diploma thesis focused on the identification of the extent of changes in the expression of Per2, Nr1d1, Stat3, BDNF genes, as well as genes encoding NMDA receptor subunits and some tissue-specific genes in the retina. Our experiments were performed on adult Long-Evans rats, that spent the first 20 days of their postnatal development in low-intensity constant light. Changes in expression were determined by quantification of mRNA by RT-qPCR in the structures of the frontal and parietal cortex, olfactory bulb, hippocampus, suprachiasmatic nucleus and retina. Behavioral tests...